In U.S. Pat. Nos. 5,593,894 and 5,989,916, incorporated herein by reference, speciation of serum cholesterol over the very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) fractions was done in a direct manner based upon a multivariate analysis treatment of visible absorbance data produced by simultaneous color derivatizations of the serum cholesterol lipoprotein subfractions. Total serum cholesterol (TC) was determined as the sum of the amounts in each fraction.
The preferred procedure, as presented in the above referenced patents, for the direct assay of cholesterol was to react a 10 xcexcL aliquot of serum with 1.0 mL of a reaction mixture comprised of an acylating compound having the general formula R.CO.R1, wherein R1 is halogen, R is selected from the group of lower alkyl radicals, and a predetermined amount of perchlorate ion effective enough to form a spectrophotometrically active product with cholesterol. The perchlorate was selected from a group consisting of barium perchlorate, zinc perchlorate, and perchloric acid. For the preferred acylating compound the R group was methyl, the R1 was chloride ion, and the preferred perchlorate was either HClO4 or Zn(ClO4)2.6H2O. Three spectral detection methods (absorbance, fluorescence, and circular dichroism) were described as being suitable for the measurement of the color intensity as a function of wavelength after a fixed time (usually 15 minutes). Absorbance detection was the preferred choice.
The choice of an acetyl chloride with either perchloric acid or zinc perchlorate hexahydrate reagent constitutes only one of many. Alternative reagent mixtures, not addressed in earlier patents, convey similar information for cholesterol and, in certain cases, additional information for other serum lipids besides cholesterol in a single simultaneous test. Alternative reagent compositions are specified in detail in this disclosure as are some options where added modifiers, in particular glacial acetic acid and acetic anhydride, can enhance the spectral data and give better control over the experimental reproducibility.
Data analyses claimed in U.S. Pat. Nos. 5,593,894 and 5,989,916 were limited to the simultaneous assays of three cholesterol variables, namely VLDL-C, LDL-C, and HDL-C, using absorbance data at only five wavelengths and the single reagent system, acetyl chloride and HClO4 catalyst. TC values were calculated as the sums of the parts.
Implications that other serum lipids (e.g. triglycerides and free fatty acids) are involved in the genesis of coronary heart disease (CHD), and appear to react with the same derivatizing reagent, stimulated the need for developing broader simultaneous assays of multiple variables but still using a single experimental procedure.
1. Objects
The object of the present invention is to expand the diagnostic capabilities of the technology from being only a serum cholesterol lipoprotein profile to a much broader serum lipids assay. The focus is on serum (or plasma) cholesterol and its analogs, and on serum (or plasma) long chain fatty acids (LCFA""s) that are present either as free acids or as esters of cholesterol, glycerol, and phospholipids. Esters are the predominant forms for LCFA""s in blood. Amounts of free acid forms of LCFA""s are, in general, very small. Taken altogether, the assay of each individual analyte would constitute a comprehensive screening of serum lipids from a single experimental test.
LCFA""s, and their ester derivatives, are both saturated and unsaturated. In natural forms the sites of unsaturation are cis-ethylenic double bonds, i.e. xe2x80x94CHxe2x95x90CHxe2x80x94. The number of double bonds ranges from one, for a series of monounsaturated fatty acids (MUFA""s), of which oleic acid is the predominant example, to polyunsaturated fatty acids (PUFA""s), represented by linoleic acid with 2 and eicosohexaenoic acid with 6 all cis- double bonds.
2. Extended Lipid Analyses
It was determined that the chromogenic acylating reagent(s), originally thought to be selective to only cholesterol, is also selective towards particular xe2x80x94CHxe2x95x90CHxe2x80x94 double bonds that are present in both cyclic and open chain aliphatic molecular structures. In serum, therefore, the actual number of lipid analytes that do react with the acylating reagent is extended to include free cholesterol (FC); saturated and unsaturated cholesteryl-LCFA esters (CE); free LCFA""s themselves; and LCFA""s in the form of triglycerides (TG) and phospholipids (PL), both of which are unsaturated LCFA esters of glycerol. Saturated LCFA""s do not react.
A red color, which is typical for standard reference materials (SRM""s) for FC and CE, is a product of reactions with the xcex945 carbonxe2x80x94carbon double bond in ring B of the steroid structure. Colors from reactions with carbonxe2x80x94carbon double bonds in SRM forms of open chain LCFA""s, in contrast, are yellow to pale orange. When SRM forms of cholesterol and LCFA are mixed and reacted in vitro, the color is an aggregate of red and yellow with the shade of orange being determined by the proportions of each component and the relative absorbance intensity of each chromophore in the products from the simultaneous acylations.
Every one of the colored products from the reactions with cholesterol analogs and PUFA analogs fluoresces. Emission fluorescence spectroscopy is an alternative detector to absorbance although less easy to use and interpret.
These same primary lipids occur in serum where they are distributed over the chylomicron, VLDL, IDL, LDL, and HDL lipoprotein fractions and subfractions, in different proportional amounts. These lipids together with apolipoproteins and other lesser components in variable amounts, determine the relative densities of the individual lipoprotein particles which is a common basis for their physical distinctions. Colors observed from acylations of synthetically prepared serum lipoprotein fractions, progress from being predominantly yellow (chylomicrons) through orange (VLDL, IDL) to pink (LDL, HDL), as the LCFA amounts decrease and the cholesterol amounts increase.
An analogous color variation is seen to occur for acylations of synthetic mimics of the Type I, IIa, IIb, III, IV, and V dyslipidemias. On the basis of quantitative interpretative analysis of the absorbance spectra associated with the resultant aggregate colors for these mixtures, the opportunity is presented to determine the amounts of each individual lipid in real serum samples with the same dyslipidemic designations.
3. Assay Procedure
The assay procedure of the present invention is performed in three general steps:
subject the lipids in a small volume aliquot of serum (plasma) to simultaneous selective acylations using a preferred color inducing reagent;
measure absorbance and/or fluorescence spectral data for the colored products over the entire visible wavelength range using a fast scanning spectrophotometer or spectrofluorimeter;
determine the amount of each lipid, and/or the proportionate amount of each lipoprotein particle in the serum lipid profile using a combination of multiple wavelength detection with modern multivariate statistical analyses methods.
4. Object of the Invention
While there have been other attempts to routinely measure total serum cholesterol and total TG in a simultaneous assay, there have been none where the focus of the assay was on either the total unsaturated lipids or the discrimination between cholesteryl and glyceryl based esters.
Although all serum lipoprotein fractions are composed from the same, relatively few, fundamental lipids, and differ only in their relative proportions in each lipoparticle, there is more than sufficient evidence from the subtle differences observed in the multiplexed spectral data to believe that a clinical diagnostic procedure has been developed that discriminates among the different serum dyslipidemias and diabetes. Its ultimate proof will depend upon the creation of a comprehensive library of spectral data for as broad a cross section of the general population as possible. The library will have sufficient number representations from normal and all forms of abnormal lipidemias and be a comprehensive resource from which can be derived more dependable, and more meaningful, risk factor models for the early assessment and prediction of coronary heart disease (CHD) and diabetes.